Use of bacterial endotoxins and lipoteichoic acids to improve postpartal health and productivity of dairy cows and their newborns

ABSTRACT

A combination of a bacterial endotoxin, in particular a lipopolysaccharide, and a lipoteichoic acid for treating or preventing a metabolic disorder or bacterial infection, or for improving milk energy efficiency in a subject. The combination may be administered separately, simultaneously or sequentially to a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority from U.S.Provisional Patent Application No. 61/448,815 filed Mar. 3, 2011, whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention concerns the use of bacterial endotoxins and lipoteichoicacids to improve postpartal health and productivity of dairy cows andtheir newborns.

BACKGROUND

The transition period is critical for the health and productivity ofdairy cows due to high incidence of metabolic disorders caused byvarious bacterial infections. Metabolic disorders are diseases thatinvolve changes in plasma metabolites of sick animals or humans. Almost50% of dairy cows are affected by one or more metabolic diseases such asketosis, fatty liver, laminitis, displaced abomasum, milk fever, downercow syndrome, udder edema, metritis, retained placenta, infertility, ormastitis. The conventional view on metabolic disorders is that thesediseases are related to the disturbance of one or more bloodmetabolites. These changes are generally interpreted as deficiencies orexcesses of these nutrients in the diet, especially, around parturition.

High-grain diets (i.e. a diet rich in starch) may be implicated in thedevelopment of metabolic disorders. Feeding ruminant animals high-graindiets is a human designed intervention to increase milk and meatproduction. However, ruminants do not naturally consume high-graindiets; rather, they eat mostly grass or forage diets. Since grain isrich in starch and poor in fiber content, feeding high-grain diets isassociated with major changes in the gastrointestinal (GI) microfloraswitching from fiber-digesting bacteria into starch-digesting bacteria.Most of the starch-digesting bacteria are Gram-negative bacteria. Thelatter degrade starch to use it for their nutritional needs. During thisprocess large quantities of acids are released into the GI tract,changing the pH from normally alkaline into acidic pH. Furthermore,abundant starch increases the number of Gram-negative bacteria in the GItract. This is associated with the release of great amounts (20-foldincrease) of toxic compounds such as endotoxin or lipopolysaccharide(LPS). Endotoxin translocates into the host's blood circulation andcauses a variety of alterations in blood metabolites, immunity, andhealth status.

Research work indicates that lipoteichoic acid (LTA) is able to inducean inflammatory response and dysfunction of multiple organs, know asseptic shock, when administered intravenously (iv) in experimentalanimals. An early investigation demonstrated that iv infusion of LTA wasassociated with the release of tumor necrosis factor alpha andinterferon gamma in the plasma, a decrease in the arterial oxygenpressure in the lungs, and increases in the plasma concentrations ofbilirubin, alanine aminotransferase, creatinine and urea, lipase frompancreas, and creatine kinase. In addition, LTA causes the release ofnitric oxide in multiple organs, circulatory failure, and 50% mortalityin the experimental animals (De Kimpe S. J. et al., 1995). Moreover,research from different groups has shown that even a single dose of LTA,as little as 0.1 mg, is sufficient to produce enhanced concentrations offree fatty acids (FFA) and triglyceride in the blood of experimentalanimals. Lipoteichoic acid also has been shown to increase theconcentration of cholesterol in the plasma. Additionally, mountingevidence indicates involvement of LTA in the pathogenesis of mastitis indairy cows. Thus, recent work demonstrated that that infusion of LTAalone in the mammary gland was sufficient to elicit a markedinflammatory response in the mammary gland of dairy cows, characterizedby a massive influx of neutrophils into milk. This suggests that duringinfection, LTA contributes to the recruitment of neutrophils

There remains a need for effective combinations and methods to improvepostpartal health and productivity of dairy cows and their newborns.

SUMMARY

In one aspect, the invention provides a combination comprising abacterial endotoxin and a lipoteichoic acid. In an embodiment, thecombination is for separate, simultaneous or sequential administrationto a subject for treating or preventing a metabolic disorder, fortreating or preventing bacterial infection or for improving milk energyefficiency in said subject.

In another aspect, the invention provides a method for treating orpreventing a metabolic disorder, for treating or preventing bacterialinfection or for improving milk energy efficiency in a subject, saidmethod comprising administering to said subject a bacterial endotoxinand a lipoteichoic acid, separately, simultaneously or sequentially.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings illustrating embodiments of the invention:

FIG. 1 is a graph depicting weekly variations of overallbeta-hydroxybutyrate in plasma of multiparous and primiparous Holsteincows challenged with oral and nasal treatment of LPS-LTA (TRT; ▪) orsaline (CTR; ⋄) (LSM±SEM; n=16; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 2 is a graph depicting weekly variations of overall non-esterifiedfatty acids in plasma of multiparous and primiparous Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=16; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 3 is a graph depicting weekly variations of overall lactate inplasma of multiparous and primiparous Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=16; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 4 is a graph depicting weekly variations of overall cholesterol inplasma of multiparous and primiparous Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=16; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 5 is a graph depicting weekly variations of overall glucose inplasma of multiparous and primiparous Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=16; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 6 is a graph depicting weekly variations of overall haptoglobin inplasma of multiparous and primiparous Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=16; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 7 is a graph depicting weekly variations of overall energycorrected milk in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=29; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 8 is a graph depicting weekly variations of overall milk efficiencyin multiparous and primiparous lactating Holstein cows challenged withoral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 9 is a graph depicting weekly variations of overall fat percent inmultiparous and primiparous lactating Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 10 is a graph depicting weekly variations of overall fat to proteinratio in multiparous and primiparous lactating Holstein cows challengedwith oral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 11 is a graph depicting weekly variations of overall fat yield inmultiparous and primiparous lactating Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=29; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 12 is a graph depicting weekly variations of overall fat correctedmilk in multiparous and primiparous lactating Holstein cows challengedwith oral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=29; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 13 is a graph depicting weekly variations of overall feed intake inmultiparous and primiparous lactating Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 14 is a graph depicting weekly variations of overall lactosecontent in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=29; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 15 is a graph depicting weekly variations of overall lactose yieldin multiparous and primiparous lactating Holstein cows challenged withoral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 16 is a graph depicting weekly variations of overall milk fatefficiency in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=30; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 17 is a graph depicting day-to-day variations of overall milkproduction in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=30; Trt=effect of treatment; Day=effect of samplingday, Trt×day=effect of treatment by sampling day).

FIG. 18 is a graph depicting weekly variations of overall milk yield inmultiparous and primiparous lactating Holstein cows challenged with oraland nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM;n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 19 is a graph depicting weekly variations of overall milk ureanitrogen in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=30; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 20 is a graph depicting weekly variations of overall milk solid notfat in multiparous and primiparous lactating Holstein cows challengedwith oral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 21 is a graph depicting weekly variations of overall milk proteincontent in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=30; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 22 is a graph depicting weekly variations of overall milk proteinyield in multiparous and primiparous lactating Holstein cows challengedwith oral and nasal treatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄)(LSM±SEM; n=30; Trt=effect of treatment; Week=effect of sampling week,Trt×Week=effect of treatment by sampling week).

FIG. 23 is a graph depicting weekly variations of overall total solidcontents in multiparous and primiparous lactating Holstein cowschallenged with oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=30; Trt=effect of treatment; Week=effect ofsampling week, Trt×Week=effect of treatment by sampling week).

FIG. 24 is a graph depicting the effect of measurement time on diurnalvariations of rumen contractions per minute in multiparous andprimiparous lactating Holstein cows challenged with oral and nasaltreatment of LPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM; n=10;Trt=effect of treatment; Time=effect of time measured before and aftertreatment, Trt×time=effect of treatment by measurement time).

FIG. 25 is a graph depicting diurnal variations of rumen contractionsper minute in multiparous and primiparous lactating Holstein cowschallenged with three different doses of oral and nasal treatment ofLPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect oftreatment; Time=effect of time measured before and after treatment,Trt×time=effect of treatment by measurement time).

FIG. 26 is a graph depicting diurnal variations of rumen contractionsper minute in multiparous and primiparous lactating Holstein cowschallenged with first dose of oral and nasal treatment of LPS-LTA (TRT;▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment;Time=effect of time measured before and after treatment, Trt×time=effectof treatment by measurement time).

FIG. 27 is a graph depicting diurnal variations of rumen contractionsper minute in multiparous and primiparous lactating Holstein cowschallenged with second dose of oral and nasal treatment of LPS-LTA (TRT;▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment;Time=effect of time measured before and after treatment, Trt×time=effectof treatment by measurement time).

FIG. 28 is a graph depicting diurnal variations of rumen contractionsper minute in multiparous and primiparous lactating Holstein cowschallenged with third dose of oral and nasal treatment of LPS-LTA (TRT;▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment;Time=effect of time measured before and after treatment, Trt×time=effectof treatment by measurement time).

FIG. 29 is a graph depicting the effect of measurement time on diurnalvariations of rectal temperature in multiparous and primiparouslactating Holstein cows challenged with oral and nasal treatment ofLPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect oftreatment; Time=effect of time measured before and after treatment,Trt×time=effect of treatment by measurement time).

FIG. 30 is a graph depicting diurnal variations of rectal temperature inmultiparous and primiparous lactating Holstein cows challenged withthree different doses of oral and nasal treatment of LPS-LTA (TRT; ▪) orsaline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect oftime measured before and after treatment, Trt×time=effect of treatmentby measurement time).

FIG. 31 is a graph depicting diurnal variations of rectal temperature inmultiparous and primiparous lactating Holstein cows challenged withfirst dose of oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect of timemeasured before and after treatment, Trt×time=effect of treatment bymeasurement time).

FIG. 32 is a graph depicting diurnal variations of rectal temperature inmultiparous and primiparous lactating Holstein cows challenged withsecond dose of oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect of timemeasured before and after treatment, Trt×time=effect of treatment bymeasurement time).

FIG. 33 is a graph depicting diurnal variations of rectal temperature inmultiparous and primiparous lactating Holstein cows challenged withthird dose of oral and nasal treatment of LPS-LTA (TRT; ▪) or saline(CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect of timemeasured before and after treatment, Trt×time=effect of treatment bymeasurement time).

FIG. 34 is a graph depicting the effect of measurement time on diurnalvariations of respiration rate per minute in multiparous and primiparouslactating Holstein cows challenged with oral and nasal treatment ofLPS-LTA (TRT; ▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect oftreatment; Time=effect of time measured before and after treatment,Trt×time=effect of treatment by measurement time).

FIG. 35 is a graph depicting diurnal variations of respiration rate perminute in multiparous and primiparous lactating Holstein cows challengedwith three different doses of oral and nasal treatment of LPS-LTA (TRT;▪) or saline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment;Time=effect of time measured before and after treatment, Trt×time=effectof treatment by measurement time).

FIG. 36 is a graph depicting diurnal variations of respiration rate perminute in multiparous and primiparous lactating Holstein cows challengedwith first dose of oral and nasal treatment of LPS-LTA (TRT; ▪) orsaline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect oftime measured before and after treatment, Trt×time=effect of treatmentby measurement time).

FIG. 37 is a graph depicting diurnal variations of respiration rate perminute in multiparous and primiparous lactating Holstein cows challengedwith second dose of oral and nasal treatment of LPS-LTA (TRT; ▪) orsaline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect oftime measured before and after treatment, Trt×time=effect of treatmentby measurement time).

FIG. 38 is a graph depicting diurnal variations of respiration rate perminute in multiparous and primiparous lactating Holstein cows challengedwith third dose of oral and nasal treatment of LPS-LTA (TRT; ▪) orsaline (CTR; ⋄) (LSM±SEM; n=10; Trt=effect of treatment; Time=effect oftime measured before and after treatment, Trt×time=effect of treatmentby measurement time).

DETAILED DESCRIPTION

Metabolic disorders are a group of diseases that affect dairy cowsimmediately after parturition. There are several metabolic disordersidentified in dairy cows during the first month after parturition, themost significant of which are the following: (1) sub-acute and acuteruminal acidosis; (2) laminitis; (3) ketosis, (4) fatty liver, (5) leftdisplaced abomasum (LDA), (6) milk fever; (7) downer cow; (8) retainedplacenta; (9) metritis, (10) mastitis, (11) udder edema; and (12) bloat.Dairy farmers lose approximately $142/cow per year for treatment ofmetabolic disorder in addition to milk loss in the first 30 days oflactation. More than half of dairy cows are affected by at least onemetabolic disorder. This makes metabolic disorders of great economicimportance.

The reason that these diseases are called metabolic disorders is relatedto the fact that they are associated with the disturbance of one or moreblood metabolites in sick cows. For example, ketosis is associated withenhanced ketone bodies in the blood; fatty liver is associated withenhanced nonesterified fatty acids (NEFA) in the blood and theiraccumulation in the liver; acidosis is associated with increasedproduction of volatile fatty acid (i.e., acetate, propionate, andbutyrate) and organic acids (e.g., lactic acid) in the rumen and lowrumen and blood pH; and milk fever is associated with decreased bloodcalcium. There is not yet a blood metabolite identified for some of themetabolic disorders such as downer cow, LDA, metritis, mastitis, orbloat. However, these diseases are associated with alteration ofmultiple blood metabolites.

The most interesting observation with regards to the occurrence ofmetabolic disorders is that they are highly associated with each other.For example, cows affected by milk fever are more prone to mastitis,retained placenta, metritis, LDA, dystocia, udder edema, and ketosis;cows affected by acidosis are more prone to laminitis, LDA, milk fever,mastitis, and fatty liver. Those affected by retained placenta are moreprone to metritis, LDA, and ketosis. Ketosis and fatty liver are commonfindings in cows affected by milk fever, mastitis, laminitis, displacedabomasum, metritis, retained placenta and udder edema. Although theseassociations have been known for years by animal scientists, the reasonbehind this association is not very well understood. One speculation isthat there might be a common etiological factor that initiates thecascade of metabolic disorders. Therefore, scientists are searching toidentify such a common causal agent of metabolic disorders; however, nosuch an agent has been identified so far.

Modern dairy cows have been selected by continuous genetic improvementand rigorous selection to achieve high milk production. Since high milkproduction cannot be maintained by forage alone grain-based diets whichare very rich in energy are fed to the cows. The ruminal digestivesystem is not developed to digest high amounts of grain and feedinggrains which are rich in starch is associated with a decline in ruminaland colonic pH, change in osmotic pressure and shift in bacterialpopulations from cellulolytic to amylolytic bacteria. Most of the knownstarch digesters are Gram-negative bacteria and this shift in populationis associated with a 20-fold increase in the amount of endotoxin in theruminal fluid. Several epidemiological studies have shown that endotoxinfrom rumen Gram-negative bacteria has been implicated in diseases thatare related to feeding high concentrate diets such as sudden deathsyndrome, ruminal acidosis, fatty liver, left displaced abomasum andlaminitis. Ruminal epithelium lacks in mucus secretion and exposure toacidotic environment leads to inflammation and tissue degeneration. Theacidotic environment, change in osmotic pressure and endotoxin mayaffect the permeability of the rumen and colon resulting intranslocation of endotoxin in the circulation. Although the presence ofendotoxin in the ruminal fluid has been documented, prior to the presentinvention there has been no convincing evidence of translocation intothe circulation.

The main objective of this investigation was to apply repeated oraladministration of lipopolysaccharide (LPS) a cell wall component ofGram-negative (GN) bacteria and lipoteichoic acid (LTA) a cell wallcomponent of Gram-positive (GP) bacteria around parturition to preventmetabolic disturbances induced by those compounds and development ofinflammatory states related to both GN and GP bacteria as well asimprove general health, and productivity of dairy cows.

Pregnant Holstein dairy cows were blocked by parity and the anticipatedday of calving, and were randomly allocated to 2 groups, 28 d before theexpected day of parturition. Cows were orally administered salinesolution (Control group), or saline solution containing 3 increasingdoses of LPS (Treatment group) form Escherichia coli 0111:B4 along witha LTA from Bacillus subtilis with the same dose pre-partum. The dose ofLTA was determined from a preliminary dosage study. Blood, urine,saliva, and vaginal mucus samples were collected 4 weeks before and 4weeks after calving, whereas milk samples were collected starting fromcalving until 4 weeks after calving for all cows in the experiment to beanalyzed for different variables. Cows were observed daily for presenceof clinical disease during the 4 weeks before and 4 weeks after calvingand rectal temperatures were taken during 3 weeks before and 2 weeksafter calving. Blood samples were also obtained from the newborns duringthe 4 weeks after birth in order to measure the immunity transmitted tothe newborn calves from the dam. Calves were also observed for incidenceof diarrhea until 4 weeks after birth.

To investigate the diurnal blood and health responses in treated cows,blood and health records were taken at −15 min before as well as 1, 3and 5 h after application of the oral vaccine. Results of this studydemonstrated that oral administration of LPS and LTA was associated withlower incidence of metritis, laminitis, retained placenta, and improveduterine horn fluctuation in the treated cows. Furthermore, the severityof laminitis was lowered in treated multiparous cows, where it tended tobe lower in the treatment group. Moreover, treated cows tended torequire lower overall number of medications as well as have lower numberof days with more than one disease versus control cows. Blood datashowed lower plasma lactate in treated cows and a tendency for higherplasma cholesterol, which is an indication of better energy status inthose cows.

Treatment did not influence plasma BHBA, NEFA, and glucose.Interestingly, data indicated that the oral vaccination of cows with LPSand LTA increased their milk energy efficiency, which was associatedwith a trend for greater feed intake in that group. Furthermore, theanalysis of milk data demonstrated a higher fat to protein ratio, aswell as greater milk fat efficiency for the treated cows. No effect oftreatment was observed on other milk components as well as on theoverall milk production. Calf data indicated a tendency for lower calfdiarrhoea score in the treatment group for both multiparous andprimiparous cows compared to controls.

Overall, administration of oral LPS and LTA improved metabolic andproductive performance as well as general health status of the treatedcows suggesting application of this novel vaccine during the transitionperiod is a very promising intervention to improve general health,productivity, and wellbeing of dairy cows and the newborns.

Endotoxin

Any bacterial endotoxin may be used in the practice of the invention.Endotoxins are cell-associated bacterial toxins. They generally composepart of the outer membrane of the cell wall of Gram-negative bacteria,whether pathogenic or not, such as Escherichia coli, Salmonella,Shigella, Pseudomonas, Neisseria, or Haemophilus. Many endotoxins arelipopolysaccharides (LPS), comprising a lipid component and apolysaccharide component. Toxicity of the endotoxin is associated withthe lipid component (lipid A) and immunogenicity is associated with thepolysaccharide component. Both lipid A and the polysaccharide componentsof LPS act as determinants of virulence in Gram-negative bacteria.

The structure of the lipid A component is highly conserved amongstGram-negative bacteria. The polysaccharide component contains tworegions. The first is known as the core (R) antigen or (R)polysaccharide. The core polysaccharide remains relatively constantwithin a bacterial genus but is structurally distinct amongst genera ofbacteria. The second polysaccharide region is the somatic (O) antigen or(O) polysaccharide. The (O) polysaccharide varies substantially betweenspecies and even amongst strains of Gram-negative bacteria.

Endotoxins of the invention may be used in purified or unpurified form.For example, in certain applications, it may be sufficient to providethe endotoxin in the form of killed bacteria, such as lysed bacteria oreven as live bacteria. In other applications, purified endotoxins (forinstance in crystalline form) may be used and are available fromcommercial sources such as Sigma-Aldrich. Synthetic endotoxins, such assynthetic LPS or LPS analogs may be used in practice of the invention.Truncated endotoxins, or portions or fractions of endotoxins comprisingonly the lipid A or core polysaccharide or (O) polysaccharide of LPS maybe used as may be chimeric endotoxins comprising an altered orheterologous lipid A or polysaccharide components.

Lipoteichoic Acid

Any lipoteichoic acid may be used in the practice of the invention.Lipoteichoic acids are a major constituent of the cell wall ofGram-positive bacteria such as Bacillus subtilis. Lipoteichoic acidsconsist of teichoic acids, long-chain ribitol phosphate and glyceridelipid membrane anchor. One function of lipoteichoic acids is asregulators of autolytic cell wall enzymes called muramidases.Lipoteichoic acids also have potent antigenic properties and canstimulate an immune response when released from bacterial cells, forinstance, after bacteriolysis induced by lysozyme, cationic peptidesfrom leucocytes, or beta-lactam antibiotics.

Lipoteichoic acids of the invention may be used in purified orunpurified form. For example, in certain applications, it may besufficient to provide the lipoteichoic acid in the form of killedbacteria, such as lysed bacteria or even as live bacteria. In otherapplications, purified lipoteichoic acids (for instance in crystallineform) may be used and are available from commercial sources such asSigma-Aldrich. Synthetic lipoteichoic acid, such as synthetic LTA or LTAanalogs may be used in practice of the invention. Truncated lipoteichoicacids, or portions or fractions of lipoteichoic acids may be used as maybe chimeric lipoteichoic acids comprising an altered or heterologousteichoic acids, ribitol phosphate chains glyceride components.

Combinations and Compositions

The endotoxin and lipoteichoic acid may be administered to a subject inthe form of, without limitation, a combination comprising a bacterialendotoxin and a lipoteichoic acid, said combination being for separate,simultaneous or sequential administration. The combinations may compriseone or more pharmaceutical compositions. Pharmaceutical compositions maybe for mucosal, oral, nasal, rectal, intravaginal or other modes ofadministration. The composition comprises the endotoxin and/or thelipoteichoic acid in combination with one or more physiologicallyacceptable ingredients, such as carriers, excipients and/or diluents.Compositions and formulations for oral administration are particularlypreferred.

Pharmaceutical compositions may be prepared, for example, in unit doseforms, such as tablets, sachets, capsules, dragees, suppositories orampoules. They may be prepared in a conventional manner, for example bymeans of conventional mixing, granulating, confectioning, dissolving orlyophilising processes.

Preferred are pharmaceutical compositions formulated for administrationto the gastrointestinal tract, such as by oral or rectal administration.Oral administration is particularly preferred as a convenient andeconomical mode of administration. Pharmaceutical compositions of thepresent invention in the form of dosage units for oral administrationmay take the form of, for example, granules, tablets, capsules, liquidsor dragees prepared together with physiologically acceptable carriers,excipients and/or diluents. Pharmaceutical compositions of the presentinvention may be applied to or incorporated into, for example, animalfeed, fodder, silage, foodstuffs and drinking water.

Typical physiologically acceptable ingredients include:

(a) binding agents such as starch (e.g. pregelatinised maize starch,wheat starch paste, rice starch paste, potato starch paste),polyvinylpyrrolidone, hydroxypropyl methylcellulose, gum tragacanthand/or gelatin;(b) fillers such as sugars (e.g. lactose, saccharose, mannitol,sorbitol), amylopectin, cellulose preparations (e.g. microcrystallinecellulose), calcium phosphates (e.g. tricalcium phosphate, calciumhydrogen phosphatelactose) and/or titanium dioxide;(c) lubricants such as stearic acid, calcium stearate, magnesiumstearate, talc, silica, silicic acid, polyethylene glycol and/or waxes;(d) disintegrants such as the above-mentioned starches, carboxymethylstarch, cross-linked polyvinylpyrrolidone, agar, alginic acid or a saltthereof (e.g. sodium alginate) and/or sodium starch glycollate;(e) wetting agents such as sodium lauryl sulphate; and/or,(f) stabilizers.

Soft gelatin capsules may be prepared with capsules containing a mixtureof the bacterial endotoxin and/or lipoteichoic acid together withparaffin oil, liquid polyethylene glycols, vegetable oil, fat and/oranother suitable vehicle for soft gelatin capsules. Plasticizers such asglycerol or sorbitol may also be used. Hard gelatin capsules may containgranules of the composition. Hard gelatin capsules may also contain theendotoxin and/or lipoteichoic acid in combination with solid powderedingredients such as those listed above.

Liquid formulations for oral administration may be prepared in the formof solutions, syrups or suspensions. Liquid formulations typicallycomprise the bacterial endotoxin and/or the lipoteichoic acid togetherwith an excipient such as sugar or sugar alcohols, and a carrier such asethanol, water, glycerol, propylene glycol, polyethylene glycol, almondoil, oily esters or mixtures thereof. If desired, such liquidformulations may also contain coloring agents, flavoring agents,saccharine, thickening agents (e.g. carboxymethyl cellulose), suspendingagents (e.g. sorbitol syrup, methyl cellulose, hydrogenated ediblefats), emulsifying agents (e.g. lecithin, acacia), and/or preservatives(e.g. methyl p-hydroxybenzoates, propyl p-hydroxybenzoates, sorbicacid). Liquid formulations for oral administration may also be preparedin the form of a dry powder to be reconstituted with water or anothersuitable vehicle prior to use.

The invention also provides kits or commercial packages comprising acomposition as described above together with printed matter comprisinginstructions for using the composition for treating or preventing ametabolic disorder, for treating or preventing bacterial infection orfor improving milk energy efficiency in a subject.

The pharmaceutical composition will generally contain a therapeuticallyeffective amount of the bacterial endotoxin and/or the lipoteichoicacid, i.e. an amount that is effective, at dosages and for periods oftime necessary, to achieve a desired prophylactic or therapeutic result,such as a reduction, inhibition, or prevention of disease onset orprogression. A therapeutically effective amount may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the compound to elicit a desired responsein the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the compound areoutweighed by the therapeutically beneficial effects.

For any particular subject, specific dosage regimens may be adjustedover time according to the individual need and the professionaljudgement of the person administering or supervising the administrationof the compositions.

In some embodiments wherein the subject is a pregnant animal such as adairy cow, the composition is administered to the subject from a time nomore than four weeks prior to parturition to a time no more than fourweeks after parturition. During this period the composition maypreferably be administered about two times per week. The composition maybe administered in a dose of from 0.01 to 1 μg endotoxin/kg body weightof the subject, more preferably from 0.01 to 0.05 μg endotoxin/kg bodyweight of the subject, even more preferably at a dose of about 0.01,about 0.05 or about 0.1 μg endotoxin/kg body weight of the subject.

In other embodiments, the composition may be administered in a dosecomprising from 0.1 to 1000 μg lipoteichoic acid, more preferably from 1to 500 μg lipoteichoic acid, more preferably from 100 to 250 μglipoteichoic acid, and even more preferably about 100, about 120 orabout 250 μg lipoteichoic acid.

Subjects

Compositions of the invention may be used in prevention of metabolicdisorders in a wide range of subjects including mammals and birds,including, without limitation: humans; livestock such as cattle, horses,goats, sheep, and pigs; companion animals such as dogs and cats; anddomesticated fowl such as chickens, ducks and geese.

In one embodiment, the subject is a ruminant mammal, such as, withoutlimitation, a cow, goat, sheep, llama, bison or deer. In an embodiment,the subject is a pregnant or has recently given birth, such as aruminant mammal within about 4 weeks before or after parturition.

Disorders

The compositions of the invention are useful for treating or preventingmetabolic disorders and bacterial infections. As used herein, “treatingor preventing” is intended to encompass curing as well as amelioratingat least one symptom of the metabolic disorder or bacterial infection,as well as obtaining beneficial or desired results including andpreferably clinical results, delaying the development or progression ofor decreasing symptoms of a metabolic disorder or bacterial infection,increasing the quality of life of those suffering from the metabolicdisorder or bacterial infection, decreasing the dose of othermedications required to treat the metabolic disorder or bacterialinfection, prolonging survival of a subject suffering from the metabolicdisorder or bacterial infection, causing the clinical symptoms of themetabolic disorder or bacterial infection not to develop byadministration of a protective composition prior to the induction ofclinical symptoms, and/or preventing recurrence of the metabolicdisorder or bacterial infection.

A metabolic disorder may be caused by or associated with parturition inthe subject and/or the feeding of a diet containing an elevatedproportion of grain-based feed or easily digestible carbohydrates. Themetabolic disorder may be associated with or caused by increasedpermeability of the colon or rumen, particularly increased permeabilitythat permits bacterial endotoxins or lipoteichoic acids to escape therumen or colon and infiltrate the bloodstream. Metabolic disorders thatmay be treated or prevented in animals, particularly ruminant mammalsinclude without limitation ruminal acidosis, laminitis, ketosis, fattyliver, left displaced abomasum, milk fever, downer cow, retainedplacenta, metritis, mastitis, udder edema or bloat. Metabolic disordersthat may be treated or prevented in humans include, without limitationabdominal obesity, impaired glucose regulation, raised triglycerides,decreased high-density lipoprotein cholesterol, elevated blood pressure,hyperinsulinemia with underlying insulin resistance, atherosclerosis,cardiovascular disease or rheumatic inflammatory disease.

Bacterial infections that may be treated or prevented include, withoutlimitation, bacterial infections caused by endotoxin-producingGram-negative bacteria, including, without limitation Escherichia coli,Salmonella, Shigella, Pseudomonas, Neisseria, or Haemophilus or bylipoteichoic acid-producing Gram-positive bacteria, including, withoutlimitation Bacillus subtilis, Staphylococcus, Streptococcus, orEnterococcus.

Improving Milk Energy Efficiency

The compositions of the invention are also useful for improving milkenergy efficiency in a subject. In one embodiment, improvement of milkenergy efficiency comprises increased fat to protein ratio. In otherembodiments, improvement of milk energy efficiency comprises increasedmilk fat efficiency. As used herein, “milk energy efficiency” (MEE) isintended to encompass the amount of milk fat in grams per kilogram ofdry matter intake, and may be calculated with the following formula:

MEE (Mcal/kg milk)=0.0929*% fat+0.0547*% Crude Protein+0.0359*% lactose.

The invention is further illustrated by the following non-limitingexamples.

Example 1

In an experiment conducted at Dairy Research and Technology Centre(DRTC; University of Alberta), thirty primiparous and multiparousHolstein dairy cows were selected for this experiment from the DairyResearch and Technology Centre (DRTC), University of Alberta.

Materials and Methods

Half of the cows were treated orally with lipopolysaccharide (LPS) fromEscherichia coli 0111:B4 and lipoteichoic acid (LTA) from Bacillussubtilis to prevent development of periparturient diseases related toLPS and LTA. At approximately 28 d before the expected day of calvingthirty pregnant primiparous and multiiparous cows were equally assignedinto two groups (n=15 per each group) including subgroups of n=10 cowsand n=5 heifers for post vaccination sampling. Based on their parity,body condition score, and milk production from previous year cows wereassigned to one of the two groups.

Cows n=15 per each group) were orally administered either 2 mL of salinesolution (Control group), or 2 mL of saline solution containing LPS fromE. coli strain 0111:B4 at three increasing concentrations as follows: 1)0.01 μg/kg BW on d −28 and −24, 2) 0.05 μg/kg BW on d −21 and −18, and0.1 μg/kg BW on d −14 along with a flat dose of LTA from Bacillussubtilis (i.e. 120 μg/animal) twice per week for 3 consecutive weeksstarting from 4 wk pre-partum (Treatment group). The initial crystallineEscherichia coli LPS (Lipopolysaccharide-FITC from E. coli strain0111:B4 purchased from Sigma-Aldrich Canada Ltd.) containing 10 mg ofpurified LPS was dissolved in 10 mL of distilled water as suggested bythe manufacturer and stored a refrigerator at +4° C. For administrationto the animal the daily dose was dissolved in 2 mL of saline and thenintroduced into the oral cavity of the cows using 5 mL disposablesyringes. Similarly, the same amount of carrier (i.e., 2 mL saline) wasorally sprayed to all cows in the control group.

All experimental procedures were approved by the University of AlbertaAnimal Care and Use Committee for Livestock, and animals were cared forin accordance with the guidelines of the Canadian Council on Animal Care(1993). Veterinary supervision was provided to the animals throughoutthe experiment.

Sampling and Analyses

Blood, saliva, vaginal mucus, and milk samples were collected twice perweek on day 1 and 3 of wk −4, −3, and −2 before the expected day ofcalving. All samples were collected before the administration ofvaccine. Blood samples from both the daily and post vaccination samplingwere analyzed for the following metabolites: beta-hydroxybutiric acid(BHBA; Wako Chemicals, Inc., Richmond, Va., USA), cholesterol(Diagnostics Chemicals Ltd., Charlottetown, PE, Canada), cortisol(Diagnostic Chemicals Ltd., Charlottetown, PE, Canada), glucose(Diagnostic Chemicals Ltd., Charlottetown, PE, Canada), non-esterifiedfatty acids (NEFA; Wako Chemicals, USA, Inc., Richmond, Va.), andinsulin (Mercodia Inc., Winston Salemm, N.C., USA). Additionally, acutephase proteins (APP) including haptoglobin (Hp; Tridelta DiagnosticsLtd, Morris Plains, N.J., USA; finished) were analyzed, whereas two moreAPP including C-reactive protein (ALPCO Diagnostics Ltd., Morris Plains,N.J., USA), and lipopolysaccharide-binding protein (HBT, Canton, Mass.,USA) will be analyzed in the near future. Plasma is analyzed foranti-LPS antibodies including immunoglobulin A (IgA), IgG, and IgM (HBTEndocab test kit HK504, Canton, Mass., USA). Plasma and milk samples aretested for content of endotoxin by the chromogenic LAL test (CapeCodeInc., MA, USA) and for LTA. Milk samples were also analyzed for fat,protein, lactose, somatic cell counts (SCC), and milk urea nitrogen(MUN) at CanWest, Dairy Herd Improvement laboratory (DHI), Edmonton,Alberta, Canada.

All treated cows were observed clinically for up to 6 h aftervaccination by measuring their rectal temperature, rumen contractionrate, and respiration rate. A dose study was conducted to determine thedose of LPS to be used for oral treatment without causing clinicalsymptoms to the animals. Blood samples from dose study were collectedseveral hours after oral treatment with LTA. Those samples were alsoanalyzed for various plasma metabolites. Disease incidence, dry matterintake (DMI), body condition score (BCS), manure score, and milkproduction records were collected for all dairy cows during 4 wk beforeand 4 wk after parturition. Reproduction records were followed untilconception or until a cull decision was taken.

Statistics

Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc.,Cary, N.C., USA Version 9.1.3) as describe by the following model:

Y _(ijkl) =m+t _(i) +w _(j) +tw _(ij) +e _(ijkl)

where Y_(ijkl) is the observations for the dependent variables, mrepresent the population mean, t_(i) is the fixed effect of treatment,w_(j) is the fixed effect of week, tw_(ij) is the interaction betweentreatment and week, and e_(ijkl) is the residual error assumed to benormally distributed. The PDIFF option of SAS was used to compare theLSM. Measurements on the same animal were considered as repeatedmeasures. The covariance structure of the repeated measurements for eachvariable was modeled separately according to the lowest values of fitstatistics based on the BIC (Bayesian information criteria). Thesignificance limit was declared at P<0.05.

Example 2

The main objective of this investigation was to apply repeated oraladministration of lipopolysaccharide (LPS) a cell wall component ofGram-negative (GN) bacteria and lipoteichoic acid (LTA) a cell wallcomponent of Gram-positive (GP) bacteria around parturition to preventmetabolic disturbances induced by those compounds and development ofinflammatory states related to both GN and GP bacteria as well asimprove general health, and productivity of dairy cows.

Thirty pregnant Holstein dairy cows were blocked by parity and theanticipated day of calving, and were randomly allocated to 2 groups(n=15 cows per group), 28 d before the expected day of parturition. Cowswere orally administered 2 mL of saline solution (Control group), or 2mL of saline solution containing 3 increasing doses of LPS (Treatmentgroup) form Escherichia coli 0111:B4 as follows: 1) 0.01 μg/kg BW on d−28 and −24, 2) 0.05 μg/kg BW on d −21 and −18, and 0.1 μg/kg BW on d−14 along with a LTA from Bacillus subtilis with the same dose (i.e. 120μg/cow) pre-partum. The dose of LTA was determined from a preliminarydosage study (see Example 3). Blood, urine, saliva, and vaginal mucussamples were collected 4 weeks before and 4 weeks after calving, whereasmilk samples were collected starting from calving until 4 weeks aftercalving for all cows in the experiment to be analyzed for differentvariables.

Cows were observed daily for presence of clinical disease during the 4weeks before and 4 weeks after calving and rectal temperatures weretaken during 3 weeks before and 2 weeks after calving. Blood sampleswere also obtained from the newborns during the 4 weeks after birth inorder to measure the immunity transmitted to the newborn calves from thedam. Calves were also observed for incidence of diarrhea until 4 weeksafter birth. To investigate the diurnal blood and health responses intreated cows, blood and health records were taken at −15 min before aswell as 1, 3 and 5 h after application of the oral vaccine.

Blood Metabolites Results

A indicated in FIG. 3, blood data showed lower plasma lactate inmultiparous and primiparous Holstein cows challenged with oral and nasaltreatment of LPS-LTA, as well as a tendency for higher plasmacholesterol (FIG. 4), which is an indication of better energy status inthose cows. Treatment did not influence plasma BHBA, NEFA, or glucose(see FIGS. 1, 2 and 5).

Milk Composition Results

Interestingly, data indicated that the oral vaccination of cows with LPSand LTA increased their milk energy efficiency (see FIGS. 7 and 8),which was associated with a trend for greater feed intake in that group(see FIG. 13).

Furthermore, the analysis of milk data demonstrated a higher fat toprotein ratio (see FIGS. 9 and 10), as well as greater milk fatefficiency for the treated cows (see FIGS. 11 and 12).

No effect of treatment was observed on other milk components as well ason the overall milk production (see FIGS. 14-23). Calf data indicated atendency for lower calf diarrhoea score in the treatment group for bothmultiparous and primiparous cows compared to controls.

Clinical Results

Results of this study demonstrated that oral administration of LPS andLTA was associated with lower incidence of metritis, laminitis, retainedplacenta, and improved uterine horn fluctuation in the treated cows (seeFIGS. 24-38). Furthermore, the severity of laminitis was lowered intreated multiparous cows, where it tended to be lower in the treatmentgroup. Moreover, treated cows tended to require lower overall number ofmedications as well as have lower number of days with more than onedisease versus control cows.

Example 3 Preliminary LTA Dosage Study

This study aimed at establishing metabolic and clinical responses toincreasing oral doses of LTA and the oral dose that will initiateclinical symptoms in dairy cows. Seven late lactating Holstein dairycows of an average BW of 800±30 kg were randomly allocated to an oraladministration of 2 mL saline solution containing one of the followingLTA doses 20, 40, 70, 100, 120, 150, and 200 μg to each cow,respectively. Blood samples were collected from the tail vein at −15min, 1, 3, and 5 h, whereas clinical responses were observed at −15 min,1, 2, 3, 4, 5, and 6 h after the oral administration of each dose ofLTA.

Blood data demonstrated that oral administration of LTA increasedconcentration of glucose in the plasma with the highest doses (150 and200 μg) having the highest plasma glucose (P<0.01). Furthermore, plasmaglucose linearly increased with time after oral administration of LTA(P<0.01). Interestingly, cows also showed greater concentrations ofplasma cholesterol at the highest doses of 150 and 200 μg (P<0.01).Also, concentrations of non-esterified fatty acid in the plasma werefound higher at 150 and 200 μg doses (P<0.01). No effect of any of thedoses of LTA used was observed on the concentration ofbeta-hydroxybutyric acid in the plasma (P>0.05). On the other hand,clinical data indicated that oral LTA influenced rectal temperatures andrespiration rates, although the variations were within the normal ranges(P<0.01 and P<0.01, respectively).

Interestingly, the highest doses of LTA (150 and 200 μg) lowered rumencontractions (P<0.01), whereas all other doses did not have an effect onthis variable. Overall, oral administration of increasing doses of LTAmodulated plasma patterns of selected metabolites and clinical responsesof late lactating dairy cows. It was also determined that the clinicalsafe dose of oral LTA to be used in future experiments was 120 μg dose.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. As used in this specificationand the appended claims, the singular forms “a”, “an”, and “the” includeplural reference unless the context clearly dictates otherwise.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A combination comprising a bacterial endotoxin and a lipoteichoicacid, said combination being for separate, simultaneous or sequentialadministration to a subject for treating or preventing a metabolicdisorder, for treating or preventing bacterial infection or forimproving milk energy efficiency in said subject.
 2. The combinationaccording to claim 1, wherein said endotoxin is a lipopolysaccharide. 3.The combination according to claim 1, wherein said bacterial endotoxinis derived from a gram-negative bacterium.
 4. The combination accordingto claim 3, wherein said gram-negative bacterium is Escherichia coli. 5.The combination according to claim 1, wherein said endotoxin is anaturally-occurring, semi-synthetic or synthetic endotoxin and whereinsaid lipoteichoic acid is a naturally-occurring, semi-synthetic orsynthetic lipoteichoic acid.
 6. The combination according to claim 1,wherein said lipoteichoic acid is derived from a gram-positivebacterium.
 7. The combination according to claim 6, wherein saidgram-positive bacterium is Bacillus subtilis.
 8. The combinationaccording to claim 1, said combination being formulated foradministration to the mucosal tissues (including gastrointestinaltract).
 9. The combination according to claim 1, said combination beingformulated for oral or nasal administration.
 10. The combinationaccording to claim 9, said combination being formulated as a tablet,capsule or liquid formulation.
 11. A method for treating or preventing ametabolic disorder, for treating or preventing bacterial infection orfor improving milk energy efficiency in a subject, said methodcomprising administering to said subject a bacterial endotoxin and alipoteichoic acid, separately, simultaneously or sequentially.
 12. Themethod according to claim 11, wherein said metabolic disorder isassociated with parturition.
 13. The method according to claim 11,wherein said metabolic disorder is metritis, laminitis, retainedplacenta or impaired uterine horn fluctuation.
 14. The method accordingto claim 11, wherein said milk energy efficiency comprises increased fatto protein ratio or increased milk fat efficiency.
 15. The methodaccording to claim 11, comprising administering said combination to saidsubject from a time no more than four weeks prior to parturition to atime no more than four weeks after parturition.
 16. The method accordingto claim 11, comprising administering said combination in an endotoxindose of from 0.001 to 1 μg endotoxin/kg body weight of said subject. 17.The method according to claim 16, wherein said endotoxin dose comprisesabout 0.01, about 0.05 or about 0.1 μg endotoxin/kg body weight of saidsubject.
 18. The method according to claim 11, comprising administeringsaid combination in a lipoteichoic acid dose of from 0.1 to 1000 μglipoteichoic acid.
 19. The method according to claim 18, wherein saidlipoteichoic acid dose comprises about 100, about 120 or about 250 μglipoteichoic acid.
 20. The method according to claim 11, wherein saidsubject is a dairy cow.